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使用氨基硅烷修饰的细菌磁性颗粒捕获和释放DNA用于单核苷酸多态性自动检测系统

Capture and release of DNA using aminosilane-modified bacterial magnetic particles for automated detection system of single nucleotide polymorphisms.

作者信息

Nakagawa Takahito, Hashimoto Reisuke, Maruyama Kohei, Tanaka Tsuyoshi, Takeyama Haruko, Matsunaga Tadashi

机构信息

Department of Biotechnology, Tokyo University of Agriculture and Technology, 2-24-16, Naka-cho, Koganei, Tokyo 184-8588, Japan.

出版信息

Biotechnol Bioeng. 2006 Aug 5;94(5):862-8. doi: 10.1002/bit.20904.

Abstract

Bacterial magnetic particles (BMPs) were modified with 3-[2-(2-aminoethylamino)-ethylamino]-propyltrimethoxysilane (AEEA) to produce a dense amine surface. Modification of BMPs in a toluene solution resulted in an increased amine yield, and approximately 11.3 x 10(4) surface amines were detected on a single particle. The modified BMPs were capable of efficient electrostatic capture of DNA. The maximum amount of DNA captured on 10 microg of aminosilane-modified BMPs was 600 ng. A 10 mM phosphate buffer effectively released the captured DNA. This efficiency was dramatically enhanced by incubation at 80 degrees C and DNA recovery from aminosilane-modified BMPs approached 95%. DNA extraction from whole blood using these modified BMPs, followed by PCR, was successfully performed. Furthermore, automated single nucleotide polymorphism (SNP) detection of the aldehyde dehydrogenase 2 (ALDH2) was demonstrated.

摘要

用3-[2-(2-氨基乙氨基)-乙氨基]-丙基三甲氧基硅烷(AEEA)对细菌磁性颗粒(BMPs)进行修饰,以产生致密的胺表面。在甲苯溶液中对BMPs进行修饰可提高胺产量,并且在单个颗粒上检测到约11.3×10⁴个表面胺。修饰后的BMPs能够有效地静电捕获DNA。10μg氨基硅烷修饰的BMPs上捕获的DNA最大量为600 ng。10 mM磷酸盐缓冲液可有效释放捕获的DNA。在80℃孵育可显著提高这种效率,从氨基硅烷修饰的BMPs中回收的DNA接近95%。使用这些修饰的BMPs从全血中提取DNA,随后进行PCR,已成功完成。此外,还展示了醛脱氢酶2(ALDH2)的自动化单核苷酸多态性(SNP)检测。

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